Abstract
Mannich bases 2a-f derived from 3,4-dimethylphenol (1), formaldehyde and different amines are prepared and subjected to spectral (IR, 1H and 13C NMR) and elemental analyses. The inhibition of two human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II, with 1 and synthesized Mannich bases 2a-f and acetazolamide (AAZ) as a control compound was investigated in vitro by using the hydratase and esterase assays. In relation to hydratase and esterase activities of the half maximal inhibitory concentration (IC50) and the inhibition equilibrium constants (Ki)values were determined. Only two compounds (2a and 2e)exhibit weak hCA II inhibitory effects on esterase activity. IC50 and Ki values for 2a and 2e with respect to esterase activity of hCA II are0.88 × 103 and 6.3–7.6 µM and 0.44 × 103 and 19.0–96.4 µM,respectively. On the contrary, compounds 2b and 2d might be used as CA activators due to increasing esterase activity of hCA I and hCA II isozymes.
Introduction
Carbonic anhydrase (CA, EC 4.2.1.1) has a catalytic function of CO2 hydration. In the active site of CA, Zn(II) ion has a tetrahedral geometry and is coordinated by the three histidine imidazoles and a water moleculeCitation1. At least 16 CA isozymes were described up to now in mammals, the most active ones as catalysts for carbon dioxide hydration being CA II and CA IXCitation2–5. Several of these isozymes (hCA II and hCA IV) are present in human eyesCitation6–9 causing glaucoma, which is a group of diseases characterized by a gradual loss of the visual field due to an elevation in intraocular pressure (IOP), and being the second leading cause of blindness worldwideCitation9,Citation10. Since CA inhibitors have been shown to reduce IOP exclusively by lowering the aqueous humour flow and these compounds have been used for the treatment of glaucoma for yearsCitation11,Citation12. The rate-determining step for the CO2 hydration reaction catalyzed by CAs is the proton transfer reaction from the water bound to the Zn(II) ion to the reaction medium, with generation of the zinc hydroxide species of the enzymeCitation13–20.
Several anions behave as inhibitors for the CO2 hydration reactionCitation21,Citation22 and coordinate directly to Zn(II) ion. A number of studies on the zinc model complexes as the active site of CA, which consist of amines and amino acid derivatives are the most investigated onesCitation23–31. The interaction of all 16 mammalian CA isozymes with several types of phenols, some of which are widely used as antioxidant food additives or as drugs has been also investigatedCitation32–35. Some antioxidant phenol derivatives have been showed effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide acetazolamideCitation36. This has given impetus for the synthesis of Mannich bases from these compounds using Mannich reaction. Mannich reaction offers a convenient method for introduction of the basic aminoalkyl chain, which alters the biological profile and physicochemical characteristicsCitation37. Various drugs obtained from Mannich reaction have proved more effective and less toxic than their parent antibioticsCitation38. In the present study, previously synthesized 2aCitation39 and novel Mannich bases 2b-f have been prepared by using microwave assist reaction of 3,4-dimethylphenol (1) with formaldehyde and various amines () which contain both functional phenolic–OH group and -NHR or -NR2 groups. They have been characterized by spectral (IR, 1H and 13C NMR) and elemental analyses. Furthermore, we have purified human CA I and II (hCA I and hCA II) from erythrocytes and examined the in vitro inhibition effects of synthesized compounds together starting compound (1) and acetazolamide (AAZ) as a control compound.
Scheme 1. Syntheses of 2a-f.
Materials and methods
All reagents were of the highest grade, commercially available and used without further purification. 1H and 13C NMR spectra were recorded with DPX-300 MHz Bruker Avance NMR spectrometer (Bio spin Gmbh, Germany). Elemental analyses for C, H and N were performed on a Leco CHNS-932 instrument. IR spectra were recorded on a Bruker Optics, vertex 70 FT-IR spectrometer using ATR techniques. A domestic microwave oven manufactured by BEKO was used for the microwave-assisted reactions at highest power (1200 W) and 2450 MHz operating frequency.
The general experimental procedure for 2a-2f
To mixture of 3,4-dimethylphenol (1) (0.1 mol) and amine compound (0.1 mol), formaldehyde (0.1 mol) was added dropwise with stirring then irradiated by microwave. After cooling to room temperature the residue was washed with ethanol and dried in air.
2-((dimethylamino)methyl)-4,5-dimethylphenol (2a)
Reaction time: 15 min; (white solid, 90%); mp 70 ˚C; 1H-NMR (d6-DMSO, ppm):8.77 (s, 1H, H1), 6.77 (s, 1H, H3), 6.51 (s, 1H, H8), 3.46 (s, 2H, H10), 2.58 (s, 3H, H7), 2.48 (s, 3H, H5), 2.10 (s, 3H, CH3), 2.07 (s, 3H, CH3); 13C-NMR (d6-DMSO, ppm): 154.20 (C2), 138.64 (C4), 131.86 (C8), 128.32 (C6), 122.43 (C3), 119.81 (C9), 59.99 (C10), 47.21 (CH3), 18.96 (C5), 18.82 (C7); IR (cm−1): 3492 (OH), 3012 (C-H)ar., 2980, 2800 (C-H)aliph., 1600-1457 (Ar C=C), 820 (ring); Anal. Calcd for C11H17NO: C, 73.70; H, 9.56; N, 7.81. Found: C, 73.86; H, 9.15; N, 7.93.
2-((diethylamino)methyl)-4,5-dimethylphenol (2b)
Reaction time: 35 min; (brown oil, 89%); 1H-NMR (d6-DMSO, ppm):9.56 (s, 1H, H1), 6.65 (s, 1H, H3), 6.44 (s, 1H, H8), 3.50 (s, 2H, H10), 2.42 (q, J = 7.03 Hz 4H, CH2), 2.20 (s, 3H, H7), 2.10 (s, 3H, H5), 0.92 (t, J = 7.01 Hz 6H, CH3); 13C-NMR (d6-DMSO, ppm): 149,53 (C2), 136.72 (C4), 129.43 (C8), 125.50 (C6), 119.52 (C3), 116.67 (C9), 55.56 (C10), 45.15 (CH2), 19.02 (C5), 18.47 (C7), 10.97 (CH3); IR (cm−1): 3485 (OH), 3006 (C-H)ar., 2961, 2873 (C-H)aliph., 1600-1440 (Ar C=C), 750 (ring); Anal. Calcd for C13H21NO: C, 75.32; H, 10.21; N, 6.76. Found: C, 75.34; H, 10.26; N, 6.81.
2-((ethyl(2-(ethylamino)ethyl)amino)methyl)-4,5-dimethylphenol (2c)
Reaction time: 60 min; (white solid, 85%); mp 120˚C;1H-NMR (d6-DMSO, ppm):10.19 (s, 1H, H1), 6.80 (s, 1H, H3), 6.49 (s, 1H, H8), 3.57 (s, 2H, H10), 2.05 (s, 3H, H7), 2.09 (s, 3H, H5), 3.34 (s, 1H, NH), 2.58(t, J = 4.71 Hz 3H, CH3), 2.50 (q, J = 4.71 Hz 2H, CH2), 2.49 (t, J = 4.68 Hz 2H, CH2), 2.48 (m, 2H, CH2), 0.99-0.89 (m, 5H, C2H5); 13C-NMR (d6-DMSO, ppm): 151.32 (C2), 141.09 (C4), 131.11 (C8), 128.42 (C6), 119.99 (C3), 119.87 (C9), 57.79 (C10), 20.01 (C5), 19.99 (C7), 55.02 (NCH2CH2), 48.67 (NCH2CH3), 46.23 (NCH2CH2NH), 44.3 (NHCH2CH3), 15.68 (NHCH2CH3), 13.42 (NCH2CH3); IR (cm−1): 3490 (OH), 2977 (NH), 3011 (C-H)ar., 2968, 2831 (C-H)aliph., 1628-1458 (Ar C=C), 748 (ring); Anal. Calcd for C15H26NO: C, 71.95; H, 10.47; N, 11.19. Found: C, 71.94; H, 10.09; N, 11.45.
4,5-dimethyl-2-(morpholinomethyl)phenol (2d)
Reaction time: 30 min; (brown oil, 92%);1H-NMR (d6-DMSO, ppm):9.82 (s, 1H, H1), 6.82 (s, 1H, H3), 6.55 (s, 1H, H8), 3.58 (s, 2H, H10), 3.59 (t, J = 4.03 Hz 4H, OCH2), 2.40 (t, J = 4.03 Hz 4H, NCH2), 2.12 (s, 3H, H7), 2.11 (s, 3H, H5); 13C-NMR (d6-DMSO, ppm): 154.89 (C2), 136.29 (C4), 130.93 (C8), 126.33 (C6), 119.34 (C3), 117.80 (C9), 66.71 (OCH2), 59.26 (C10), 53.17 (NCH2), 19.63 (C5), 18.79 (C7). IR (cm−1); 3489 (OH), 3010 (C-H)ar., 2990, 2907 (C-H)aliph., 1635-1448 (Ar C=C), 750 (ring); Anal. Calcd for C13H19NO2: C, 70.56; H, 8.65; N, 6.33. Found: C, 70.62; H, 8.66; N, 6.37.
2-((dibenzylamino)methyl)-4,5-dimethylphenol (2e)
Reaction time: 35 min; (white solid, 96%); mp 96 ˚C; 1H-NMR (d6-DMSO, ppm):10.41 (s, 1H, H1), 7.28-7.87 (m, 10H, C6H5), 6.67 (s, 1H, H3), 6.66 (s, 1H, H8), 3.69 (s, 2H, H10), 3.62 (s, 4H, CH2), 2.20 (s, 3H, H7), 2.17 (s, 3H, H5); 13C-NMR (d6-DMSO, ppm): 153.62 (C2), 137.92 (C4), 137.46–126.98 (C6H5-CH), 132.94 (C8), 129.85 (C6), 119.85 (C3), 119.74 (C9), 63.56 (C6H5-C), 55.84 (C10), 19.20 (C5), 18.80 (C7); IR (cm−1): 3495 (OH), 3015 (C-H)ar., 2950, 2850 (C-H)aliph., 1560-1455 (Ar C=C), 736 (ring); Anal. Calcd for C23H25NO: C, 83.34; H, 7.60; N, 4.23. Found: C, 83.36; H, 7.41; N, 4.35.
2-((4-fluorophenylamino)methyl)-4,5-dimethylphenol (2f)
Reaction time: 20 min; (white solid, 82%); mp 145˚C;1H-NMR (d6-DMSO, ppm):9.13 (s, 1H, H1), 6.90 (s, 1H, H3), 6.92 (s, 1H, H8), 6.50 (m, 2H, C6H4), 6.40 (m, 2H, C6H4), 5.82 (s, 1H, NH), 4.06 (s, 2H, H10), 2.08 (s, 3H, H7), 2.04 (s, 3H, H5); 13C-NMR (d6-DMSO, ppm): 156.68 (C6H4-C), 151.02 (C2), 145.01 (C6H4-C), 138.52 (C4), 130.00 (C8), 129.01 (C6), 126.13 (C3), 120.01 (C9), 119.13 (C6H4-CH), 53.42 (C10), 116.52 (C6H4-CH), 21.01 (C5), 19.68 (C7); IR (cm−1): 3488 (OH), 3276 (NH), 3007 (C-H)ar., 2976, 2884 (C-H)aliph., 1502-1453 (Ar C=C), 763 (ring); Anal. Calcd for C15H16FNO: C, 73.45; H, 6.57; N, 5.71. Found: C, 73.36; H, 6.37; N, 5.85.
Purification of isoenzymes hCA-I and hCA-II from human erythrocytes
In order to purify hCA-I and hCA-II isoenzymes, first, human blood was centrifuged at 1500 rpm for 20 min, and after the removal of the plasma, the erythrocytes were washed with an isotonic solution (0.9% NaCl). After that, the erythrocytes were lysed with 1.5 volume of ice-cold water. The lysate was centrifuged at 20,000 rpm for 30 min to remove cell membranes and non-lysed cells. The pH of the supernatant was adjusted to 8.7 with tris and was then loaded onto an affinity column containing Sepharose-4B-L-tyrosine-p-aminobenzene sulfonamide as the binding group. After extensive washing with 25 mM tris–HCl/22 mM Na2SO4 (pH 8.7), the hCA-I and hCA-II isoenzymes were eluted with 1.0 M NaCl/25 mM Na2HPO4 (pH 6.3) and 0.1 M CH3COONa/0.5 M NaClO4 (pH 5.6)Citation40,Citation41. The amount of purified protein was estimated by the Bradford methodCitation42 and SDS–PAGE was carried out to determine whether the elute containing the enzymeCitation43.
Hydratase activity assay
Carbonic anhydrase activity was assayed by following the hydration of CO2 according to the method described by Wilbur and AndersonCitation44. CO2-hydratase activity as an enzyme unit (EU) was calculated by using the equation ((t0-tc)/tc) where t0 and tc are the times for pH change of the nonenzymatic and the enzymatic reactions, respectively. IC50 values (the concentration of inhibitor producing a 50% inhibition of CA activity) have been obtained as in vitro for the synthesized compounds 2a-f, 1 and AAZ as the control compound.
Esterase activity assay
Carbonic anhydrase activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer (CHEBIOS UV–VIS) according to the method described in the literatureCitation45,Citation46. The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL of 0.05 M tris–SO4 buffer (pH 7.4), 1 mL of 3 mM 4-nitrophenylacetate, 0.5 mL H2O and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution. IC50 values have been obtained as in vitro for free the synthesized compounds 2a-f, 1 and AAZ as the control compound.
Determination of Ki values
The method for determination of Ki values is described elsewhereCitation47–51. In the first part of this study, IC50 values have been obtained as in vitro. IC50 of the inhibitors (the synthesized compounds 2a-f, 1 and AAZ as the control compound) were assayed by the hydrolysis of p-nitrophenylacetate on esterase activities of CA isoenzymes in the presence of various inhibitor concentrations. The absorbance was determined at 348 nm after 3 minCitation47. Regression analysis graphs were drawn by plotting inhibitor concentrations versus enzyme activity by using Microsoft Excel Program.
In the second part of the study, enzyme activity was measured in the presence of five different substrate concentrations at each of these inhibitor concentrations (30%, 50%, and 70%), and the data were linearized with Lineweaver–Burk plot in order to obtain Ki values.
Result and discussion
We herein report the syntheses of title compounds 2a-f by treatment of 3,4-dimethylphenol (1), with various amines in the presence of formaldehyde. The reactions were carried out under microwave (MW) irradiation and solvent free condition. The one-step Mannich reaction of 2a-f is given in .
1H-NMR and 13C-NMR spectra of 2a-f
The 1H and 13C-NMR spectra of compounds 2a-f were recorded in d6-DMSO solution at room temperature using TMS as internal standard. In the 1H NMR spectra the signals of the respective protons of the prepared compounds 2a-f were verified on the basis of their chemical shifts, multiplicities and coupling constants. The NMR spectra of 2a-f exhibited a broad singlet in the range 10.41-8.77 ppm which correspond to protons H1 (O-H)Citation40. The chemical shift of the aromatic protons of the benzene ring are observed in the range 6.90-6.65 ppm for H3 protons and 6.92-6.44 ppm for H8 protons. The 1H NMR spectra of 2a-f showed a singlet with 2 H intensity each, in the range 4.06-3.46 ppm assigned to the H10 (CH2) protons, confirming the Mannich condensation of 3,4-dimetylphenol, formaldehyde and aminesCitation52,Citation53. In the 1H NMR spectra of compounds 2a-f showed two singlets with 3 H intensity each, in the range 2.58-2.05 and 2.48-2.04 ppm for protons H7 and H5, respectively.
13C NMR spectra of 2a-f exhibited peaks in the range 154.89–149.53, 141.09-136.29, 132.94–129.43, 129.85–125.50, 126.13–119.34 and 120.01-116.67 ppm due to C2, C4, C8, C6, C3 and C9 carbon atoms, respectively. The carbon atoms (C10) for 2a-f were observed in the range 59.99-53.42 ppm. The signals in the range 21.01-18.96 and 19.99-18.47 are assigned to C5 and C7 carbon atoms, respectively.
FTIR measurements
The IR spectra of all compounds showed vibrational bands in the range 3495-3485 cm−1 for O-H group. The bands at 3276 and 2977 cm−1 are assigned to vibrations associated with the N-H moiety for compounds 2f and 2c, respectivelyCitation54. Aromatic C-H stretching vibrations for all compounds are observed in the range 3015-3006 cm−1. The bands in the range 2990-2950 and 2907-2800 cm−1 are attributable to aliphatic C-H stretching vibrations for compounds 2a-f. The IR spectra of all compounds showed vibrational bands in the range 1635-1440 cm−1 for C=C groupCitation55. The bands of ring are located in the range 820-736 cm−1.
In vitro inhibition studies
Inhibition effects on hCA I and hCA II isozymes of the synthesized compounds (2a-f) and acetazolamide (AAZ) as the control compound were studied by hydratase and esterase activity methods. The related Ki values were also determined for each compound in order to compare with inhibition effects of starting compound (1) and AAZ ().
Table 1. IC50 and Ki values forsynthesized compounds (2a-f), 1 and AAZ withhCA I and hCA II isozymes.
According to in vitro studies, there is no inhibition effects observed for 1 and synthesized compounds (2a-f) on hydratase activities of hCA I and hCA II isozymes, and for 2c and 2f on esterase activities of hCA I and hCA II isozymes. Starting compound 1 showed esterase activities on hCA I and hCA II. However, IC50 values for the esterase activities of 1 (1.03 × 103 µM for hCA I and 0.93 × 103 µM for hCA II, respectively) were greater than the IC50 values of AAZ (5.9 µM for hCA I and 4.3 µM for hCA II, respectively). In relation to esterase activities, the inhibition equilibrium constants (Ki) were also determined. Compound 1 exhibited Ki values as 8.5-79.6 µM for hCA I and 15.3-67.3 µM for hCA II, respectively indicating poorer inhibition effects compared to AAZ (5.0-6.1 µM for hCA I and 3.5-4.1 µM for hCA II, respectively).
Compounds 2a and 2e showed inhibitory effects on esterase activity of hCA II, nevertheless they did not showed any inhibitory effects of hCA I isozyme. The esterase IC50 and the Ki values for 2a and 2e (0.88 × 103 and 6.3–7.6 µM for hCA II, respectively and 0.44 × 103 and 19.0-96.4 µM for hCA II, respectively) are higher than the esterase IC50 and the Ki values of AAZ (4.3 and 3.5-4.1 µM for hCA II, respectively). Similar to 1, these compounds are also weaker inhibitors than AAZ. No inhibition effects were observed for compounds 2b and 2d on hCA I and hCA II isozymes but these compounds increased the esterase activities of these isozymes. Therefore, 2b and 2d compounds might be evaluated as carbonic anhydrase activators.
The compounds used in this study have both functional phenolic -OH group and -NHR or -NR2 groups together in a molecule which have not reinforced their CA (I and II) inhibitory effects. This might be due to acid-base reaction between them, although they are good CA inhibitors separatelyCitation23–28,Citation30,Citation32,Citation36.
Conclusion
An efficient, clean, economic, and one-pot procedure for the synthesis of Mannich bases (2a-f) has been used by the three-component coupling of aldehyde, amine, and 3,4-dimethylphenol (1) under microwave irradiation, solvent free conditions, short reaction time with excellent yields of the products. All of the synthesized compounds 2a-f have been completely characterized by IR, 1H and 13C NMR and elemental analyses and all results are consistent with the proposed structures (). Inhibition effects of synthesized compounds (2a-f)on hCA I and hCA II isozymes were studied by hydratase and esterase activity methods and then Ki values were determined. None of the synthesized compounds (2a-f) did not show any inhibition effect on hydratase activity of hCA I and hCA II. Only 2a and 2e showed weak inhibitory effects on esterase activity of hCA II. On the contrary, compounds 2b and 2d might be good CA activators due to increasing esterase activity of hCA I and hCA II isozymes.
Declaration of interest
This work was supported by grant (Grant No: 2008-1) from the Dumlupınar University Research Foundation and carried out in the Department of Chemistry of the Dumlupınar University.
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